Occupational hypersensitivity pneumonitis (HP) in machine workers has been associated with mycobacteria that frequently colonize modern water-based metalworking fluids (MWFs). HP is an immune-mediated lung disease caused by inhalation of microbial antigens and is relatively poorly studied in terms of its diagnosis, pathogenesis, and treatment. Overall goal of this study is to identify and characterize antigens associated with HP-inducing potential of MWF mycobacteria species M. immunogenum (MI) and M. chelonae (MC) and evaluate their potential for HP immunodiagnosis and exposure assessment. In previous funding cycles, we have isolated multiple strains (genotypes) of both the species with variable antigenic characteristics. We will test our hypothesis that antigen diversity in MWF mycobacteria strains prevalent in commercial MWF is responsible for differentially inducing the HP symptoms and underlying immune response, and that the responsible specific antigens could form the basis for more informative diagnosis and personal and environmental exposure assessment. The specific aims are to: (1). Identify key antigens in MI and MC strains based on their relative interaction with the host immune system in an optimized mouse model of HP;(2). Recombinant production of the identified key antigens by cloning and protein expression and generation of specific antibodies;(3). Investigate potential of the specific antigens for early personal exposure assessment and immunodiagnosis in symptomatic versus asymptomatic individuals;(4). Evaluate potential of the specific antigen(s) of MWF mycobacteria for environmental exposure assessment (fluid monitoring). Relative immunogenicity of the isolated different genotypes of MI (6) and MC (4) will be compared based on HP induction potential and cell-mediated immune response using our optimized mouse model and the putative protein antigens will be identified by differential immunoproteomic profiling. The recombinantly expressed antigens will be evaluated for their potential for immunodiagnosis using the mouse model and human subjects, particularly in terms of differentiation of the asymptomatic (exposed) versus symptomatic (HP patients) and for environmental exposure assessment. The resulting information will help NORA's objectives by providing a set of tools for mycobacterial exposure assessment and HP immunodiagnosis;these outcomes will facilitate future epidemiological studies and development of prevention (vaccination) and intervention strategies for MWF exposures in long-term. PUBLIC HEALTH RELEVANCE: Non-tuberculous mycobacteria (NTM) colonizing modern metalworking fluids (MWFs) have been associated with occupational respiratory illnesses. Recent studies by NIOSH and other groups have linked MWF mycobacteria with occupational hypersensitivity pneumonitis (HP) in exposed machine workers. HP is an immune-mediated lung disease caused by inhalation of microbial antigens. Hence, characterization of specific antigens in MWF mycobacteria and evaluation of their potential for HP immunodiagnosis and exposure assessment will facilitate development of pre-emptive measures and intervention strategies in MWF-associated occupational environments.